Prolyl Endoprotease Research Articles

Production of the prolyl endoprotease (PEP) from Aspergillus sp. FSDE 16 by solid-state fermentation (SSF) and use for producing a gluten-free beer.

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17±5.31 and 21.19±11.28ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

In vitro Gluten Degradation Using Recombinant Eurygaster Integriceps Prolyl Endoprotease: Implications for Celiac Disease.

Celiac disease (CD) is a gluten-sensitive chronic autoimmune enteropathy. A strict life-long gluten-free diet is the only efficient and accepted treatment until now. However, maintaining a truly gluten-free status is both difficult and costly, often resulting in a social burden for the person. Moreover, 2 to 5 percent of patients fail to improve clinically and histologically upon elimination of dietary gluten. Therefore, novel therapeutic approaches, including gluten degrading enzymes, are an unmet need of celiac patients. To evaluate the function of sunn pest prolyl endoprotease for gluten and gliadin hydrolysis in vitro. The spPEP was expressed as a recombinant protein in E. coli BL21 (DE3), and its catalytic activity was assessed by SDS-PAGE and RP-HPLC analyses. Production of a 100-kDa spPEP protein was confirmed by SDS-PAGE and western blot analysis. Also, we demonstrate that spPEP efficiently degrades gluten and α-gliadin (30-40 kDa) in vitro under conditions similar to the GI and is resistant to pepsin and trypsin. The gathered data demonstrated that spPEP might be a novel candidate for Oral Enzymatic Therapy (OET) in CD and other gluten-related disorders.

Characterization of gluten-degrading prolyl endoprotease from Thermococcus kodakarensis.

There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14mM/s, 1.10mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Targeted degradation of gluten proteins in wheat flour by prolyl endoprotease and its utilization in low immunogenic pasta for gluten sensitivity population

Gluten is responsible for adverse immune effects and causes wheat allergy, celiac disease, and other wheat-related disorders to genetically predisposed individuals. Wheat flour protein immunogenicity can be reduced by proteolytic degradation enzymes. The present study aims to utilize Aspergillus niger prolyl endoprotease (EC 3.4.21.26) to specifically cleave the proline-rich sequences and degradation of gluten proteins in the wheat flour. The Triticum aestivum (HD-2851, NIAW-917), Triticum durum (UAS-428), Triticum dicoccum (DDK- 1025) were taken for proteolytic degradation using AN-PEP. The enzymatic modification was carried out in an optimum condition (pH-4.0, at 40 °C) for 5 h. The wheat flour gliadin content was reduced up to 90–95% in HD-2851, DDK-1025, NIAW-917 and UAS-428 shown up to 74% reduction of gliadins. The complete hydrolysis of gliadins (α, β, ω, ϒ) and glutenin subunits were confirmed by using immunoblot, ELISA, RP-HPLC chromatograph, anti-PEP-I (PSQQQQQPK), anti-PEP-II (LGQQQPFPPQQPYPQPQPFK) and anti-HMW-GS antibodies. The modified wheat flour was taken for the processing of pasta with the replacement of 30% chickpea flour. The gluten content of MWF pasta was found to be 48 ppm which is 99.95% lesser than the control pasta. Sensory evaluation revealed that the pasta was acceptable with overall quality when compared with control pasta.

A randomised, double-blind, cross-over trial to evaluate bread, in which gluten has been pre-digested by prolyl endoprotease treatment, in subjects self-reporting benefits of adopting a gluten-free or low-gluten diet.

The aim of the present study was to determine if the enzyme Aspergillus niger prolyl endoprotease (ANPEP), which degrades the immunogenic proline-rich residues in gluten peptides, can be used in the development of new wheat products, suitable for gluten-sensitive (GS) individuals. We have carried out a double-blind, randomised, cross-over trial with two groups of adults; subjects, self-reporting benefits of adopting a gluten-free or low-gluten diet (GS, n 16) and a control non-GS group (n 12). For the trial, volunteers consumed four wheat breads: normal bread, bread treated with 0·8 or 1 % ANPEP and low-protein bread made from biscuit flour. Compared with controls, GS subjects had a favourable cardiovascular lipid profile - lower LDL (4·0 (sem 0·3) v. 2·8 (sem 0·2) mmol/l; P=0·008) and LDL:HDL ratio (3·2 (sem 0·4) v. 1·8 (sem 0·2); P=0·005) and modified haematological profile. The majority of the GS subjects followed a low-gluten lifestyle, which helps to reduce the gastrointestinal (GI) symptoms severity. The low-gluten lifestyle does not have any effect on the quality of life, fatigue or mental state of this population. Consumption of normal wheat bread increased GI symptoms in GS subjects compared with their habitual diet. ANPEP lowered the immunogenic gluten in the treated bread by approximately 40 %. However, when compared with the control bread for inducing GI symptoms, no treatment effects were apparent. ANPEP can be applied in the production of bread with taste, texture and appearance comparable with standard bread.

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

Randomized clinical trial: Effective gluten degradation by Aspergillus niger-derived enzyme in a complex meal setting

The Aspergillus niger-derived prolyl endoprotease (AN-PEP) has previously been shown to degrade gluten in healthy subjects when added to an intragastrically infused meal. The current study investigated the efficacy of AN-PEP in a physiological meal setting. In this randomized placebo-controlled crossover study, 18 gluten-sensitive subjects consumed a porridge containing 0.5 g gluten together with two tablets either containing a high or low dose of AN-PEP, or placebo. Gastric and duodenal content was sampled over 180 minutes, and areas under the curve of gluten concentrations were calculated. The primary outcome, i.e. success rate of high dose AN-PEP defined as at least 50% gluten degradation compared to placebo in the duodenum, was achieved in 10 of 13 comparisons. In the stomach, gluten levels were reduced from 176.9 (median, interquartile range 73.5–357.8) to 22.0 (10.6–50.8, p = 0.001) in the high dose and to 25.4 μg × min/ml (16.4–43.7, p = 0.001) in the low dose. In the duodenum, gluten levels were reduced from 14.1 (8.3–124.7) in the placebo to 6.3 (3.5–19.8, p = 0.019) in the high dose and to 7.4 μg × min/ml in the low dose (3.8–12.0, p = 0.015). Thus even in a physiological meal setting, AN-PEP significantly degraded most gluten in the stomach before it entered the duodenum.

Discovery, cloning and characterisation of proline specific prolyl endopeptidase, a gluten degrading thermo-stable enzyme from Sphaerobacter thermophiles

Gluten free products have emerged during the last decades, as a result of a growing public concern and technological advancements allowing gluten reduction in food products. One approach is to use gluten degrading enzymes, typically at low or ambient temperatures, whereas many food production processes occur at elevated temperature. We present in this paper, the discovery, cloning and characterisation of a novel recombinant thermostable gluten degrading enzyme, a proline specific prolyl endoprotease (PEP) from Sphaerobacter thermophiles. The molecular mass of the prolyl endopeptidase was estimated to be 77kDa by using SDS-PAGE. Enzyme activity assays with a synthetic dipeptide Z-Gly-Pro-p-nitroanilide as the substrate revealed that the enzyme had optimal activity at pH 6.6 and was most active from pH 5.0-8.0. The optimum temperature was 63 °C and residual activity after one hour incubation at 63 °C was higher than 75 %. The enzyme was activated and stabilized by Co2+ and inhibited by Mg2+, K+ and Ca2+ followed by Zn2+, Na+, Mn2+, Al3+, and Cu2+. The Km and kcat values of the purified enzyme for different substrates were evaluated. The ability to degrade immunogenic gluten peptides (PQPQLPYPQPQLPY (a-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein)) was also confirmed by enzymatic assays and mass spectrometric analysis of cleavage fragments. Addition of the enzyme during small scale mashing of barley malt reduced the gluten content. The findings here demonstrate the potential of enzyme use during mashing to produce gluten free beer, and provide new insights into the effects of proline specific proteases on gluten degradation.

Reduction of Hordein Content in Beer by Applying Prolyl Endoprotease to the Malting Process

Barley malt contains hordein proteins, which gluten-sensitive consumers cannot tolerate. Beer produced from barley malt also contains hordeins. Aspergillus niger prolyl endoprotease (AN-PEP) is an enzyme that has been used effectively to reduce beer hordeins during fermentation. The objective of this study was to apply AN-PEP during the steeping and germination of barley and evaluate the impact on malt quality characteristics and the hordein content of model beers. Pilot-scale malting trials were performed, and the barley was germinated for either 3 or 5 days with and without AN-PEP. Model beers were produced from malts, and the levels of beer hordeins were tested using an R5 antibody-based competitive enzyme-linked immunosorbent assay. The malt friability, extract, viscosity, and several other quality parameters were measured following industry-standard MEBAK methods. Treatment of malt with AN-PEP for 5 days resulted in a 46% reduction in beer hordeins compared with beer produced from the 5 day control malt, and the quality of the AN-PEP treated malt was comparable to untreated malt. Applying enzymes to germinating grain is a novel way to influence the levels of hordeins in barley malt beers.

Neprosin, a Selective Prolyl Endoprotease for Bottom-up Proteomics and Histone Mapping

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

Inhibition of Eurygaster Integriceps Puton Prolyl Endoprotease (spPEP) and Human Prolyl Endopeptidase (hPEP) using αS1 ‐ Casein Peptide Inhibitors

Prolyl endopeptidases (PEPs) are widely distributed among all domains of life (Di Cera., 2009). The focus of this study is mainly on prolyl endoprotease originated from the salivary glands of Erygaster integriceps Puton (Sunn pest) identified as spPEP by Darkoh (2008, 2010). The widely spread Eurygaster genus represents the most damaging and highly adaptable wheat pest in Northern Africa, Eastern Europe, Middle East with increasing concerns in Australia and United States. The natural substrate for spPEP is gluten from wheat grains, making spPEP the first known eukaryotic PEP capable of degrading substrates longer than 30 amino acid residues. To study the binding mechanism of this unique enzyme, recombinant peptide inhibitors were generated from αS1‐casein containing the inhibitory sequence LNENLLRFFVAPFPEV (Juillerat‐Jeanneret et al., 2011) varying in the length of the peptide and the positioning of the inhibitory sequence. All peptides were constructed with an N‐terminal 6x His Tag for purification as well as a Thrombin protease site for His Tag cleavage, ligated into pET15b vector and transformed into BL21(DE3)pLysS expression host (Vishram.,2016). The expression and purification of the peptides, including thrombin digestion and HPLC for final purification and quantification are shown. Enzyme kinetics for each of the recombinant inhibitory peptides in the presence of GPpNA as substrate is compared between spPEP and human prolyl endopeptidase (hPEP). This is the first study to show the hPEP to bind a peptide longer than 30 amino acid residues as well as inhibition of spPEP with large peptides.

Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers.

SummaryBackground Aspergillus niger prolyl endoprotease (AN‐PEP) efficiently degrades gluten molecules into non‐immunogenic peptides in vitro.AimTo assess the efficacy of AN‐PEP on gluten degradation in a low and high calorie meal in healthy subjects.MethodsIn this randomised, double‐blind, placebo‐controlled, cross‐over study 12 healthy volunteers attended to four test days. A liquid low or high calorie meal (4 g gluten) with AN‐PEP or placebo was administered into the stomach. Via a triple‐lumen catheter gastric and duodenal aspirates were sampled, and polyethylene glycol (PEG)‐3350 was continuously infused. Acetaminophen in the meals tracked gastric emptying time. Gastric and duodenal samples were used to calculate 240‐min area under the curve (AUC 0–240 min) of ?‐gliadin concentrations. Absolute ?‐gliadin AUC 0–240 min was calculated using duodenal PEG‐3350 concentrations.Results AN‐PEP lowered α‐gliadin concentration AUC 0–240 min, compared to placebo, from low and high calorie meals in stomach (low: 35 vs. 389 μg × min/mL; high: 53 vs. 386 μg × min/mL; P < 0.001) and duodenum (low: 7 vs. 168 μg × min/mL; high: 4 vs. 32 μg × min/mL; P < 0.001) and absolute α‐gliadin AUC 0–240 min in the duodenum from low (2813 vs. 31 952 μg × min; P < 0.001) and high (2553 vs. 13 095 μg × min; P = 0.013) calorie meals. In the placebo group, the high compared to low calorie meal slowed gastric emptying and lowered the duodenal α‐gliadin concentration AUC 0–240 min (32 vs. 168 μg × min/mL; P = 0.001).ConclusionsAN‐PEP significantly enhanced gluten digestion in the stomach of healthy volunteers. Increasing caloric density prolonged gastric residence time of the meal. Since AN‐PEP already degraded most gluten from low calorie meals, no incremental effect was observed by increasing meal caloric density. ClinicalTrials.gov, Number: NCT01335503; www.trialregister.nl, Number: NTR2780.

Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

Use of Pseudomonas aeruginosa Chaperones in Folding an Insect Recombinant Prolyl Endoprotease (rPEP) Expressed in Escherichia coli.

Chaperone assist in protein folding. The purpose of this study were to 1) determine if co‐expression of Pseudomonas aeruginosa GroESL or Trigger factor with prolyl endo protease (PEP) cDNA in Escherichia coli system resulted in increase amount of soluble active enzyme 2) determine if two different temperatures, 37°C for 24 hours and biphasic temperature incubation of 37°C for 6 hours followed by overnight in 28°C would increase in amount of soluble active PEP and 3) to identify which chaperone mechanism results in highest expression of active protein. GroESL and TF chaperones were cloned into pLys‐S and pLys‐E plasmid. The resulting pGROELS‐S/E plasmid or pTF‐S/E plasmid was co‐transformed into E. coli. Western Blot techniques were used to determine the level of protein expression. Enzyme activity was assayed using a synthetic chromagenic peptide. Biphasic temperature growth resulted in higher level of active than constant 37°C. pGroESL‐ E and pTF‐S resulted in highest increase in amount of soluble rPEP as compared to amount found. Expression in presence of pGroESL‐E and pTF‐S resulted in the highest levels of active soluble enzyme. P. aeruginosa GroESL and TF chaperones co‐express and assist in folding successfully expression of a large insect PEP in E. coli.Funding: Office of Research and Sponsored Projects, Stephen F Austin State University; Ed and Gwen Cole, Nacogdoches, TX.

Use of Recombinent Casein Peptides as Inhibitors of spPEP

Eurygaster integreceps (Sunn pest) causes major damage in wheat grains by injecting their saliva into the grain degrading the gluten proteins. This saliva protein has been identified and characterized as a prolyl endoprotease (spPEP) in our lab (Darkoh et al, 2010). A natural, biological friendly inhibitor to the spPEP is being sought. Previous studies have shown that casein peptides generated by Lactobacillis species have inhibitory effects on mammalian and bacterial prolyl endoproteases (PEP). The Objective of this study was to clone the four bovine casein genes, α1, α2, β and κ for use in expressing each isomer to identify which peptides inhibit the spPEP. The nucleotide sequences corresponding to bovine casein isomers were identified from the NCBI database to design gene specific primers that would amplify the full length casein proteins. Total RNA was extracted from bovine mammary gland biopsy tissue kindly donated by Dr. Erin Connor, Research Leader, USDA‐ARS, Beltsville, MD and subsequent cDNA has been synthesized. Each casein subunit was cloned into the pGEM T‐Easy vector for sequencing. Verified sequences were cloned into the pET15b E.coli expression vector for protein expression and purification using IMAC chromatography. Purified proteins will be used for subsequent incubation with L. helveticus at varying times. The resulting peptide mixture will be incubated with the spPEP in the presence of the GPpNA substrate to determine which isomer/s contains the spPEP inhibitory sequences.Funding Sources: Office of Research and Sponsored Projects, Stephen F. Austin State University, Nacogdoches, TX and Ed and Gwen Cole, Nacogdoches, TX.

Consumption of gluten with gluten-degrading enzyme by celiac patients: A pilot-study

To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients. Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint. In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits. AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Celiac Disease: Advances in Treatment via Gluten Modification

Celiac disease (CD) is an autoimmune enteropathy that occurs in genetically susceptible individuals carrying the prerequisite genetic markers HLA DQ2 or DQ8. These genetic markers are present in approximately 30% of the population, and the worldwide prevalence of CD is estimated to be approximately 1%-2%. Currently a gluten-free diet is the only treatment for CD, but novel therapies aimed at gluten modification are underway. This review will discuss gluten-based therapies including wheat alternatives and wheat selection, enzymatic alteration of wheat, oral enzyme supplements, and polymeric binders as exciting new therapies for treatment of CD.